Population structures in the SARA and SARB reference collections of Salmonella enterica according to MLST, MLEE and microarray hybridization

In the 1980's and 1990's, population genetic analyses based on Multilocus Enzyme Electrophoresis (MLEE) provided an initial overview of the genetic diversity of multiple bacterial species, including Salmonella enterica. The genetic diversity within S. enterica subspecies enterica according to MLEE is represented by the SARA and SARB reference collections, each consisting of 72 isolates, which have been extensively used for comparative analyses. MLEE has subsequently been replaced by Multilocus Sequence Typing (MLST). Our initial MLST results indicated that some strains within the SARB collection differed from their published descriptions. We therefore performed MLST on four versions of the SARB collection from different sources and one collection of SARA, and found that multiple isolates in SARB and SARA differ in serovar from their original description, and other SARB isolates differed between different sources. Comparisons with a global MLST database allowed a plausible reconstruction of the serovars of the original collection. MLEE, MLST and microarrays were largely concordant at recognizing closely related strains. MLST was particularly effective at recognizing discrete population genetic groupings while the two other methods provided hints of higher order relationships. However, quantitative pair-wise phylogenetic distances differed considerably between all three methods. Our results provide a translation dictionary from MLEE to MLST for the extant SARA and SARB collections which can facilitate genomic comparisons based on archival insights from MLEE

Involvement of the Rcs regulon in the persistence of Salmonella Typhimurium in tomatoes

It is becoming clear that human enteric pathogens, like Salmonella, can efficiently colonize vegetative and reproductive organs of plants. Even though the bacterium's ability to proliferate within plant tissues has been linked to outbreaks of salmonellosis, little is known about regulatory and physiological adaptations of Salmonella, or other human pathogens, to their persistence in plants. A screen of Salmonella deletion mutants in tomatoes identified rcsA and rcsB genes as those under positive selection. In tomato fruits, populations of Salmonella rcsB mutants were as much as 100-fold lower than those of the wild type. In the follow-up experiments, competitive fitness of rcsA and rcsB mutants was strongly reduced in tomatoes. Bioinformatics predictions identified a putative Salmonella RcsAB binding box (TTMGGAWWAABCTYA) and revealed an extensive putative RcsAB regulon, of which many members were differentially fit within tomatoes

Genome-wide analysis of the PreA/PreB (QseB/QseC) regulon of Salmonella enterica serovar Typhimurium

<p>Abstract</p> <p>Background</p> <p>The <it>Salmonella </it>PreA/PreB two-component system (TCS) is an ortholog of the QseBC TCS of <it>Escherichia coli</it>. In both <it>Salmonella </it>and <it>E. coli</it>, this system has been shown to affect motility and virulence in response to quorum-sensing and hormonal signals, and to affect the transcription of the <it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium) <it>pmrAB </it>operon, which encodes an important virulence-associated TCS.</p> <p>Results</p> <p>To determine the PreA/PreB regulon in <it>S</it>. Typhimurium, we performed DNA microarrays comparing the wild type strain and various <it>preA </it>and/or <it>preB </it>mutants in the presence of ectopically expressed <it>preA </it>(<it>qseB</it>). These data confirmed our previous findings of the negative effect of PreB on PreA gene regulation and identified candidate PreA-regulated genes. A proportion of the activated loci were previously identified as PmrA-activated genes (<it>yibD</it>, <it>pmrAB</it>, <it>cptA</it>, etc.) or were genes located in the local region around <it>preA</it>, including the <it>preAB </it>operon. The transcriptional units were defined in this local region by RT-PCR, suggesting three PreA activated operons composed of <it>preA-preB</it>, <it>mdaB-ygiN</it>, and <it>ygiW</it>-STM3175. Several putative virulence-related phenotypes were examined for <it>preAB </it>mutants, resulting in the observation of a host cell invasion and slight virulence defect of a <it>preAB </it>mutant. Contrary to previous reports on this TCS, we were unable to show a PreA/PreB-dependent effect of the quorum-sensing signal AI-2 or of epinephrine on <it>S</it>. Typhimurium with regard to bacterial motility.</p> <p>Conclusion</p> <p>This work further characterizes this unorthadox OmpR/EnvZ class TCS and provides novel candidate regulated genes for further study. This first in-depth study of the PreA/PreB regulatory system phenotypes and regulation suggests significant comparative differences to the reported function of the orthologous QseB/QseC in <it>E. coli</it>.</p

Genome-wide analysis of the PreA/PreB (QseB/QseC) regulon of Salmonella enterica serovar Typhimurium

Background. The Salmonella PreA/PreB two-component system (TCS) is an ortholog of the QseBC TCS of Escherichia coli. In both Salmonella and E. coli, this system has been shown to affect motility and virulence in response to quorum-sensing and hormonal signals, and to affect the transcription of the Salmonella enterica serovar Typhimurium (S. Typhimurium) pmrAB operon, which encodes an important virulence-associated TCS. Results. To determine the PreA/PreB regulon in S. Typhimurium, we performed DNA microarrays comparing the wild type strain and various preA and/or preB mutants in the presence of ectopically expressed preA (qseB). These data confirmed our previous findings of the negative effect of PreB on PreA gene regulation and identified candidate PreA-regulated genes. A proportion of the activated loci were previously identified as PmrA-activated genes (yibD, pmrAB, cptA, etc.) or were genes located in the local region around preA, including the preAB operon. The transcriptional units were defined in this local region by RT-PCR, suggesting three PreA activated operons composed of preA-preB, mdaB-ygiN, and ygiW-STM3175. Several putative virulence-related phenotypes were examined for preAB mutants, resulting in the observation of a host cell invasion and slight virulence defect of a preAB mutant. Contrary to previous reports on this TCS, we were unable to show a PreA/PreB-dependent effect of the quorum-sensing signal AI-2 or of epinephrine on S. Typhimurium with regard to bacterial motility. Conclusion. This work further characterizes this unorthadox OmpR/EnvZ class TCS and provides novel candidate regulated genes for further study. This first in-depth study of the PreA/PreB regulatory system phenotypes and regulation suggests significant comparative differences to the reported function of the orthologous QseB/QseC in E. coli

Novel two-step hierarchical screening of mutant pools reveals mutants under selection in chicks

© 2016, American Society for Microbiology. All Rights Reserved.Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection

Infection of Mice by Salmonella enterica Serovar Enteritidis Involves Additional Genes That Are Absent in the Genome of Serovar Typhimurium

Salmonella enterica serovar Enteritidis causes a systemic, typhoid-like infection in newly hatched poultry and mice. In the present study, a library of 54,000 transposon mutants of S. Enteritidis phage type 4 (PT4) strain P125109 was screened for mutants deficient in the in vivo colonization of the BALB/c mouse model using a microarray-based negative-selection screening. Mutants in genes known to contribute to systemic infection (e.g., Salmonella pathogenicity island 2 [SPI-2], aro, rfa, rfb, phoP, and phoQ) and enteric infection (e.g., SPI-1 and SPI-5) in this and other Salmonella serovars displayed colonization defects in our assay. In addition, a strong attenuation was observed for mutants in genes and genomic islands that are not present in S. Typhimurium or in most other Salmonella serovars. These genes include a type I restriction/modification system (SEN4290 to SEN4292), the peg fimbrial operon (SEN2144A to SEN2145B), a putative pathogenicity island (SEN1970 to SEN1999), and a type VI secretion system remnant SEN1001, encoding a hypothetical protein containing a lysin motif (LysM) domain associated with peptidoglycan binding. Proliferation defects for mutants in these individual genes and in exemplar genes for each of these clusters were confirmed in competitive infections with wild-type S. Enteritidis. A ΔSEN1001 mutant was defective for survival within RAW264.7 murine macrophages in vitro. Complementation assays directly linked the SEN1001 gene to phenotypes observed in vivo and in vitro. The genes identified here may perform novel virulence functions not characterized in previous Salmonella models

Spaceflight Alters Bacterial Gene Expression and Virulence and Reveals Role for Global Regulator Hfq

A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the spaceflight environment has never been accomplished due to significant technological and logistical hurdles. Moreover, the effects of spaceflight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared to identical ground control cultures. Global microarray and proteomic analyses revealed 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground based microgravity culture model. Spaceflight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during spaceflight missions and provide novel therapeutic options on Earth

Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation

Using sample-matched transcriptomics and proteomics measurements it is now possible to begin to understand the impact of post-transcriptional regulatory programs in Enterobacteria. In bacteria post-transcriptional regulation is mediated by relatively few identified RNA-binding protein factors including CsrA, Hfq and SmpB. A mutation in any one of these three genes, csrA, hfq, and smpB, in Salmonella is attenuated for mouse virulence and unable to survive in macrophages. CsrA has a clearly defined specificity based on binding to a specific mRNA sequence to inhibit translation. However, the proteins regulated by Hfq and SmpB are not as clearly defined. Previous work identified proteins regulated by hfq using purification of the RNA-protein complex with direct sequencing of the bound RNAs and found binding to a surprisingly large number of transcripts. In this report we have used global proteomics to directly identify proteins regulated by Hfq or SmpB by comparing protein abundance in the parent and isogenic hfq or smpB mutant. From these same samples we also prepared RNA for microarray analysis to determine if alteration of protein expression was mediated post-transcriptionally. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all possible Salmonella proteins, respectively, with limited correlation between transcription and protein expression. These proteins represent a broad spectrum of Salmonella proteins required for many biological processes including host cell invasion, motility, central metabolism, LPS biosynthesis, two-component regulatory systems, and fatty acid metabolism. Our results represent one of the first global analyses of post-transcriptional regulons in any organism and suggest that regulation at the translational level is widespread and plays an important role in virulence regulation and environmental adaptation for Salmonella